Immunological analysis of food proteins using high-performance thin-layer chromatography-immunostaining

Abstract

The chromatographic analysis of intact proteins is still challenging, especially when biological functions as antigenicity of proteins or peptides are in the focus. Traditional immunoassays provide information about the entirety of antigenic proteins/peptides, e.g., in ELISA assays. On the other hand, when focusing on the investigation of (cross) reactivity of antibodies, Western blot following gel-electrophoresis represents the method of choice. However, gel-electrophoresis is limited by the molecular weight and therefore, not suitable for peptides ≤3kDa or proteins ≥250kDa. Furthermore, for gaining detailed information about the protein sequence (e.g., via mass spectrometric analysis), a so called in-gel digest needs to be performed following electrophoretic separation and is therefore elaborate and accompanied by a significant loss of structural, and even more severe, conformational information. Here, protein analysis using HPTLC seems to be a promising alternative due to the high level of variability regarding the chromatographic system (multiple mobile and stationary phases, even mixed) and manifold detection as well as hyphenation possibilities.This study exemplarily focused on the immunological investigation of proteins in milk following thin-layer chromatographic separation. The detection of these antigens is mandatory, as they might trigger allergenic reactions in sensitized people. Besides the proof of its applicability on different stationary phase materials, the newly developed immunoassay can be used as an approach for semi-quantitative estimation of antigenic proteins. In addition to the analysis of intact food allergens, also analyzing peptides thereof is worth considering which can be realized using HPTLC-immunostaining as well.